Opioids have been used very successfully for the treatment of moderate to severe acute and chronic pain. Unfortunately, their uses have been associated with many troublesome side effects such as nausea, constipation, respiratory depression, sedation, pruritus, tolerance and dependence development. Many approaches have been used to alleviate these side effects without diminishing the analgesic effects, with variable success. Notable approaches have been the design of receptor selective ligands that would activate one of the three cloned opioid receptors and co-administration of pharmaceutical agents to block the opioid side effects. Although these approaches and others are viable ones, one of the reasons for their limited success is the high amino acid sequence homology among the cloned receptors. Such homology has slowed the design and development of an opioid drug that will target and activate a specific opioid receptor. In the current studies, we propose to use an alternative approach, i.e., to engineer mutant opioid receptors for pain management. Our approach is based on an accidentally discovered receptor mutation, S4.45(196)L in the u-opioid receptor (MOR), that resulted in the ability of opioid alkaloid antagonists to activate the mutant receptor, both in vitro and in vivo. We hypothesize that, if such a mutant receptor could be delivered to and expressed in the nociceptive neurons, then activation of the mutant receptors by antagonists should produce analgesic responses without eliciting the tolerance responses during chronic treatment with the antagonists. Our overall goal is to develop such receptor mutants as therapeutic agents for pain management. Thus, in the current proposal, we will (1) determine the molecular bases for the activation of receptor mutants by opioid antagonists;(2) validate and refine the receptor model for S4.54 mutation so as to engineer a mutant MOR in which naloxone and naltrexone behave like full agonists;and (3) develop a double stranded adenoassociated virus (dsAAV) for the delivery of the mutant receptor at specific sites of the pain pathway and evaluate the antagonist and agonist efficacies in eliciting antinociceptive responses. We will examine both the acute and chronic responses to opioid agonists and antagonists after dsAAV injection. By developing the receptor model and demonstrating the structural bases for the antagonist activities via receptor mutagenesis studies and generation of mutant mouse lines, we could engineer a mutant receptor in which opioid antagonists behave like full agonists. Since dsAAV has been used successfully in gene therapy, the eventual delivery by dsAAV and expression of the mutant receptor at the nociceptive neurons that normally express MOR should result in analgesic responses after systemic administration of opioid antagonists without tolerance development. Such a mutant opioid receptor gene therapy approach could be a new paradigm for the eventual treatment of chronic pain. PUBLIC HEALTH RELEVANCE: In the treatment of moderate to severe pain, morphine remains the drug of choice. However, the many side-effects of the drugs, notably tolerance and dependence development in prolonged treatment, have reduced the desirability in the use of this opioid analgesic for pain management. It has been the Holy Grail of pharmacologists and pharmaceutical chemists to develop drug molecules or treatment paradigms that elicit the pain relief effects without any side effects. With the discovery of a mutant u-opioid receptor (MOR) that could be activated by opioid antagonists without altering the agonists'properties, we hypothesize that such a mutant receptor could be developed into therapeutic agents for the purpose of pain management. We have demonstrated the feasibility of such approach by "knocking-in" this mutation, S4.54(196)A, into MOR, and generating a mouse line in which opioid antagonists, naloxone and naltrexone produced antinociceptive responses. However, this mutation only resulted in partial agonistic properties observed with these two opioid antagonists. Therefore, in the proposed studies, we will investigate the molecular bases for such antagonistic activities using modeling and mutational analysis. Additional receptor mutations will be identified in order to convert the antagonists into full agonists. We will develop a gene therapy vehicle, initially using dsAAV2 virus, to deliver the mutant receptors into various regions of the pain pathway and to examine the feasibility of using opioid antagonists as antinociceptive agents. The activation of the exogenously introduced mutant MOR, and the inactivation of the endogenous opioid receptors by the antagonists, will provide a unique opportunity to develop a pain treatment paradigm without possible development of tolerance and dependence.